Capilarry pins for high-efficiency microarray printing device

ABSTRACT

This invention provides improved components (e.g. array “pins”, print head, substrate platen, print head platen, and the like) for microarray printing devices as well as microarray printing devices incorporating such components. In one embodiment, this invention provides a microarray print head comprising a plurality of glass or quartz spotting capillaries disposed in a support that maintains a fixed spacing between the spotting capillaries and that permits the spotting capillaries to move in a direction parallel to the long axis of the capillaries.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 09/894,863,filed on Jun. 27, 2001, which is incorporated herein by reference in itsentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] [Not Applicable]

FIELD OF THE INVENTION

[0003] This invention pertains to the field of high-density microarrayproduction. In particular, this invention provides methods and devicesthat permit high-density arrays to be printed with significantly smallerfeature size and spacing and greatly improved reagent usage.

BACKGROUND OF THE INVENTION

[0004] The immobilization of test molecules or “probes” on arraysupports has had a significant impact on drug discovery, medicaldiagnostic methods, and basic research. The use of high-densitymicroarrays of organic molecules permits literally thousands of assaysto be simultaneously performed on one or more samples. Usinghigh-density microarrays, numerous analytes can be simultaneouslydetected and/or quantified permitting the rapid characterization ofcomplex systems (e.g. complex assays for gene expression). High-densitymicroarrays are also useful for “high-throughput” screening assays,diagnostics, and in many other contexts. The ability to manufacturemicroarrays in an efficient and cost-effective manner is of considerableinterest to researchers worldwide and of significant commercial value.

[0005] In general, microarrays of greater density are preferred. Ahigher density array typically allows more assays to be performedsimultaneously and/or, for lower sample volumes to be used for the samenumber of assays. In providing large, high-density arrays of molecules(e.g., probes or analytes) there are a number of considerations. Thearray elements (e.g. dots) should be substantially reproducible in size,particularly if one wishes to quantify an analyte. In addition, thearray elements should be consistently and reliably positioned, andshould be highly reproducible.

[0006] The basic approaches for generating arrays of test molecules suchas nucleic acid, protein or other organic molecules fall into twogeneral categories. In the first such approach the test molecules aredirectly synthesized onto the array support, while in the second suchapproach the test molecules are attached to the supportpost-synthetically. Each approach has its own limitations and drawbacks.For example, when an array is created by direct synthesis onto an arraysupport, the efficiency of each synthetic step affects the quality andintegrity of molecules forming the array. The magnitude of the problemincreases with the complexity of the individual molecules, potentiallyresulting in an undesirable percentage of incorrectly synthesizedmolecules and incomplete sequences. Such contaminants can interfere withsubsequent use of the array.

[0007] In addition, synthetic approaches (e.g. as described by Southernet al. (U.S. Pat. Nos. 5,770,367, 5,700,637, and 5,436,327), Pirrung etal. (U.S. Pat. No. 5,143,854), Fodor et al. (U.S. Pat. Nos. 5,744,305and 5,800,992), and Winkler et al. (U.S. Pat. No. 5,384,261), aregenerally unable to construct microarrays of large macromolecules. Suchtechnologies can also be expensive and difficult to implement.

[0008] In contrast, the second approach to array production allows thedesired molecules to be produced (e.g. synthesized, isolated, amplified,e.g.) by conventional methods prior to their formation into an array.Consequently, the quality of the arrayed molecules, and thus the qualityof the resultant array, is potentially greater than that produced by thedirect synthesis approach.

[0009] Such “spotting” approaches include, but are not limited toinkjet, and direct surface contact printing. Inkjet devices require highreagent volumes and risk “probe” degradation during volatilization.

[0010] Direct surface contact printing (see, e.g., U.S. Pat. Nos.4,981,783, 5,525,464, 5,770,151, and 5,807,522), are limited in theirability to reliably, reproducibly, and uniformly apply the arrayelements to the array substrate. Reagent usage is also relativelyinefficient, and array density is limited.

SUMMARY OF THE INVENTION

[0011] The present invention provides improved components (e.g. array“pins”, print head, substrate platen, print head platen, and the like)for microarray printing devices as well as microarray printing devicesincorporating such components. In particular, methods and devices ofthis invention permit high-density arrays to be printed withsignificantly smaller feature size and spacing, and greatlyimproved-reagent usage.

[0012] In one embodiment this invention provides a microarray printhead, said print head comprising a plurality of glass or quartz (orother mineral), or ceramic, or porcelain, or ceramic spottingcapillaries disposed in a support that maintains a fixed spacing betweenthe spotting capillaries and that permits the spotting capillaries tomove in a direction parallel to the long axis of the capillaries (i.e.the spotting capillaries can slide through the support). Preferredspotting capillaries are microcapillary tubes and particularly preferredspotting capillaries have a tapered tip (e.g. a ground, beveled tip).The capillaries can have any desirable cross-section (e.g. round, ovoid,square, triangular, irregular), however preferred capillaries are roundin cross-section.

[0013] In certain preferred embodiments, the capillaries have a maximumload volume of about 0.5 mL. In certain preferred embodiments, thespotting capillaries have a load volume of about 0.2 mL.

[0014] Preferred print heads comprise at least 4 spotting capillaries,preferably least , 4, 16, 32, 64, or 128 spotting capillaries and incertain preferred embodiments, the spacing between two adjacent spottingcapillaries is about 3 mm or less, center to center.

[0015] In certain preferred embodiments, the spotting capillaries havedetents where the spotting capillaries have a rest position in which thedetents contact a support stopping the movement of the spottingcapillaries in a direction toward the substrate that is to be printed.The print head can also comprise a spring attached to a spottingcapillary where, in the absence of a force against the printing tip ofthe spotting capillary the spring returns said spotting capillary to arest position. The print head can be provided separately or can be foundas a component in a microarray printing device.

[0016] In preferred embodiments, the spotting capillaries are in fluidcommunication (e.g. via flexible capillary tubing) with a manifold. Inpreferred embodiments, the manifold comprises a common port and aplurality of individual ports where an aperture into an individual portis disposed inward of the inside wall of the manifold. The manifold canbe connected to a gas and/or vacuum source.

[0017] In another embodiment this invention provides a platen forpositioning a substrate holder or a print head in a microarray printingdevice. A preferred platen comprises a support surface attached to asingle guide rail such that the support surface can move along the guiderail, and motion of the support is constrained in a direction normal tothe guide rail, and a flexible coupling to an actuator wherein theflexible coupling is rigid or stiff in a direction parallel to the guiderail, but is flexible in another direction. The platen also, optionally,comprises an encoder (e.g. optical encoder, magnetic encoder, electronicencoder, etc.) that encodes the position of said platen along said guiderail. In certain embodiments, the platen is attached to the rail by twobearings. Preferred flexible couplings include, but are not limited to aflexible sheet coupling (e.g. sheet metal, sheet plastic, etc.), a rodbearing, a ball bearing, a pin bearing and the like. Preferred actuatorsinclude, but are not limited to a stepping motor, a linear motor, a leadscrew, and the like. In certain embodiments, the platen can furthercomprise a holder (e.g. a slide holder) for one or more microarraysubstrates. In certain embodiments, the platen is attached to amicroarray print head (e.g. directly or through a movable stage).Preferred print heads in such cases include, but are not limited to anyof the print heads described herein.

[0018] In still another embodiment, this invention provides a microarrayprinting device comprising a microarray print head (e.g. as describedherein); and a microarray substrate holder attached to a platen (e.g. aplaten as described herein). Preferred microarray printers can print atleast 2,000, more preferably at least 5,000 array elements per spottingcapillary per load. Preferred microarray printers can print arrayelements with a precision of at least 30 μm and/or with an averageinter-element spacing of 130 μm or less. Preferred microarray printerscan print 200 or more microarray substrates in a run. Particularlypreferred microarray printers of this invention utilize pressure and/orvacuum to control reagent loading or dispensing. Certain microarrayprinters comprise the spotting capillaries are in fluid communicationwith a manifold. A preferred manifold comprises a common port andindividual ports where an aperture into an individual port is disposedinward of the inside wall of the manifold. In certain preferredembodiments, the microarray printing device can loads reagents from amicrotiter plate comprising at least about 864 wells.

[0019] This invention also provides a method of printing a microarray(e.g., a nucleic acid and/or protein and/or small organic moleculemicroarray). The methods involve providing an array substrate in amicroarray printing device comprising one or more of the elements (e.g.spotting capillaries, print head, array substrate platen, print headplaten, and the like) as described herein, providing a series ofsolutions comprising the reagents that will form features of themicroarray; and operating said microarray printing device to print themicroarray. In preferred methods the microarray printing device prints amicroarray comprising at least 1,000 different array elements. Inpreferred methods the microarray printing device prints a microarraycomprising having an average inter-feature spacing of about 130 μm orless. Preferred array substrates include, glass, quartz or otherminerals, metals, ceramics, plastics, metal coated glass, metal coatedplastic and the like. In preferred methods the microarray printingdevice applies negative pressure to load a spotting capillary and/orpositive pressure to dispense from a spotting capillary. In preferredembodiments, the method involves loading feature-forming reagents from amicrotiter plate comprising at least about 864 wells.

[0020] In still another embodiment, this invention provides (printed)microarrays. Preferred printed microarrays comprise at least about 1000different array elements on an array substrate, where the array elementsare separated by an average center to center spacing of about 130 μm orless, preferably about 100 μm or less, more preferably about 80 μm orless. Where the arrays are nucleic acid and/or protein arrays, theprotein or said nucleic acid is preferably not a chemically synthesizedprotein or nucleic acid. Particularly preferred microarrays includenucleic acid nucleic acid microarrays. In certain embodiments, thenucleic acids comprising such microarrays have an average length greaterthan about 50, preferably greater than about 100, 200, or 500nucleotides, more preferably greater than about 1000 nucleotides. Themolecules comprising the array features are preferably adsorbed to thearray substrate. In certain nucleic acid or protein arrays the nucleicacid or protein is not covalently coupled to the array substrate (e.g.not coupled directly or though a linker to a terminal nucleotide oramino acid). In particularly preferred microarrays, the featurescomprising the arrays are at an average density of about 40,000/cm² orgreater.

DEFINITIONS

[0021] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide.

[0022] The terms “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein refer to at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention is preferablysingle-stranded or double stranded and will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800;Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986)Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805,Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al.(1986) Chemica Scripta 26: 1419), phosphorothioate (Mag et al. (1991)Nucleic Acids Res. 19:1437; and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321,O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc.114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen(1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Otheranalog nucleic acids include those with positive backbones (Denpcy etal. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger etal. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994)Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & MedicinalChem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17;Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev.pp169-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of additional moietiessuch as labels, or to increase the stability and half-life of suchmolecules in physiological environments.

[0023] The terms “spotting capillary” and “pin” or “printing pin” areused synonymously to refer to the structure that is used to contact amicroarray substrate and thereby deposit a reagent to form a microarrayfeature on that substrate. Unlike many printing pins, however, thespotting capillary is typically a tube and, while not limited to such,in certain preferred embodiments, display a round cross section.

[0024] An “array substrate” refers to the surface or support on which amicroarray is printed. Array substrates include, but are not limited toglass, quartz or other minerals, ceramic, porcelain, metal, andmetal-coated glass.

[0025] An “array feature” or “array spot” refers to a reagent orreagents deposited at a location on an array surface. Typically afeature is characterized by the presence of one or more specificmolecules (e.g. particular proteins, nucleic acids, etc.).

[0026] A “guide rail” refers to a rail or other device that directs ororients the movement of a platen as described herein. In certainembodiments, guide rail can take any of a number of forms including, butnot limited to T-shaped, round, triangular, square, ovoid, and the like.The guide rail is typically coupled to the platen through one or morebearings that permit motion of the platen in one direction (along oneaxis), but restrict motion in other directions. In certain preferredembodiments, the guide rail is configured to to control the motion eachdegree of freedom of the spotter (e.g. the platten and/or print head)the parts of the robot with the minimal number of restraints that arerequired by Euclidian geometry, e.g. as described herein.

[0027] A “print cycle” refers to the sequence of events involved inprinting an array feature.

[0028] The term “microarray” refers to an array comprising at leastabout 10, preferably at least about 50, more preferably at least about100, still more preferably at least about 500 or 1000, and mostpreferably at least about 10,000, 40,000, 100,000, or 1,000,000different and distinct features. Preferred microarrays have an averagefeature density greater than about 100/cm², more preferably greater thanabout 1000/cm², still more preferably greater than about 5,000/cm², evenstill more preferably greater than about 10,000/cm², and most preferablygreater than about 20,000/cm², 40,000/cm², 60,000/cm², or even 80,000cm².

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIGS. 1A and 1B illustrates a microarray printer print head 10 ofthis invention. FIG. 1A illustrates a print head 10 comprising a seriesof guide plates 14 that support and position the spotting capillaries12. The downward motion of the spotting capillaries is limited by adetent 20 and the spotting capillaries are returned to the “extended”position by a spring 18 compressed between the detent and a springcapture plate 22. The spotting capillaries communicate to a manifoldthrough a flexible capillary tubing 24. FIG. 1B illustrates a print headcapable of mounting 64 spotting capillaries on 3 mm centers for 864 wellmicrotiter plates. In this illustration, 16 spotting capillaries are inuse.

[0030]FIG. 2 illustrates a glass or quartz spotting capillary of thisinvention showing the inside diameter 32, the outside diameter 34, andthe bevel 30 at the tip.

[0031]FIG. 3 illustrates a two-rail positioning platen compared to apreferred one-rail platen.

[0032]FIG. 4 illustrates one embodiment of a platen used to position anarray substrate in an array printing device.

[0033]FIG. 5 illustrates an exploded view of a platen used to positionan array substrate in an array printing device.

[0034]FIG. 6 illustrates one embodiment of a platen used to position amicroarray print head in an array printing device.

[0035]FIG. 7 schematically illustrates vacuum and pressure plumbing of amicroarray printer of this invention.

[0036]FIG. 8 illustrates a preferred manifold design.

[0037]FIG. 9 illustrates the print head and its associated plumbing.

[0038]FIG. 10 illustrates unidirectional flow through a manifoldaccording to the methods and devices of this invention. The manifold 64is connected to flexible tubing 84 a leading to a vacuum source and toflexible tubing 84 b leading to a pressure source. Flow isunidirectional in the direction of the open arrow.

[0039]FIG. 11 schematically illustrates controls for the pressure sideof the pressure control system for printhead operation.

[0040]FIG. 12 schematically illustrates controls for the vacuum side ofthe pressure control system for printhead operation.

[0041]FIG. 13 illustrates a scheme for passive magnetic control ofprinting pin/capillary position.

[0042]FIG. 14 illustrates a scheme for active magnetic control ofprinting pin/capillary position.

DETAILED DESCRIPTION

[0043] This invention pertains to a microarray printer that can be usedto manufacture microarrays (e.g. of biochemical samples) by directcontact printing. The array printer of this invention is capable ofprinting microarrays at higher feature density, with greater speed andlower cost than previous microarray printing devices.

[0044] Without being bound to a particular theory, these efficienciesare achieved by the use of a combination of novel features. A novelprinting pin permits vastly more efficient reagent usage and a greaternumber features to be printed per load. This reduces reagent costs, andbecause the pin does not require repeated refills during the printingprocess, the printing operation proceeds more rapidly.

[0045] In addition, positive control of reagent flow using pressure andvacuum, also improves reagent capture and delivery and reduces theincidents of mis-prints due to pin blockage during loads or print steps.In addition, positive pressure control keeps the liquid at the tip ofthe printing capillary. This significantly improves printingreliability.

[0046] A novel design for the substrate support permits the use of alarger positioning substrate that can hold a greater number of arraysubstrates (e.g. more than 10, preferably more than 20 or 50, still morepreferably greater than 100, and most preferably greater than 150, 200,250, 300, or even greater than about 500 standard slide-sizedsubstrates) and position each array with greater accuracy and precision.The substrate support is kinematically constrained so that printingsubstrates are more reproducibly positioned even at rapid accelerationsand decelerations, thereby thereby permitting a print run to proceedwith greater rapidity, i.e., decreasing effective print time andreducing printing costs, and to print arrays with a higher density ofspots.

[0047] A related kinematic support design for the print head permitrapid acceleration and deceleration of the print head and reproduciblepositioning. The rapid precise positioning of the array substratecombined with the rapid positioning of the print head again,significantly reduces the time required for a print cycle therebyreducing costs, and increases the utility of the arrays by allowingproduction of a higher density of spots.

[0048] These features combine to make possible the efficient printing ofmicroarrays at extremely high efficiency with low reagent usage, andpreviously unobtainable feature spacing for printed microarrays.

[0049] I. Printing Pins and the Print Head.

[0050] In one embodiment, this invention provides for print head 10 forprinting microarrays and a microarray printer comprising such a printhead. As illustrated in FIG. 1, in one preferred embodiment, the printhead 10 comprises a plurality of spotting capillaries 12 disposed in asupport 14 that maintains a fixed spacing between the spottingcapillaries and that permits the spotting capillaries to move in adirection parallel to the long axis of said capillaries (i.e., thespotting capillaries can slide in the support).

[0051] The spotting capillaries 12 are preferably cylinders (e.g.capillary tubes) made of a rigid material such as glass, quartz or othermineral, ceramic, brittle plastic (e.g. acrylic), and the like. It was asurprising discovery of this invention that glass-like materials such asglass or quartz or ceramic could be effectively used as spottingcapillaries. Moreover, particularly when fabricated and utilized asdescribed herein, such glass, quartz or ceramic spotting capillarieshave a useful lifetime vastly greater than that observed for thecommonly utilized metal pins. Indeed, we have yet to determine themaximum lifetime of the spotting capillaries described herein, while itis believed that metal spotting are quite limited in their useful life.

[0052] The spotting capillaries used in this invention can be of anyconvenient size, however, in preferred embodiments (see, e.g., FIG. 2),the spotting capillaries are microcapillaries, with an inside diameter(bore width) at the tip of less than about 100 μm, preferably less thanabout 75 μm, more preferably less than about 50 μm, and most preferablyless than about 30 μm, 25 μm or even less than about 20 μm. The lowersize limit (bore diameter) can be determined by practical considerationsof plugging given the occurrence of particulate matter in somepreparations. Thus, there can be practical size limits on the printingcapillary tip opening size given the care (cleanliness) with which theprinting solutions are prepared. If the inside diameter of the capillaryis too small and the capillary is too long, then the flow resistances ofthe capillary can impede the ability to rapidly draw cleaning solutionsetc. through it. Thus, in certain preferred embodiments, the capillariesare used that have a larger ID away from the tip, and the capillarydiameter (ID and OD) is reduced at the tip to produce a small feature(spot) size. The load volume (loaded fluid volume) of the spottingcapillary is typically 1 μL or less, preferably 0.5 μL or less, morepreferably 0.25 μL or less, and most preferably 0.2 μL or less or even0.1 μL or less.

[0053] As indicated above, the spotting capillaries need not have aconstant internal diameter. The diameter (ID and especially OD) at theaperture (spotting face) of the spotting capillary will, in part,determine the minimum feature size of the spotted microarray. Thus,smaller aperture and external tip diameters are preferred. Particularlypreferred aperture diameters (ID or OD) are less than about 75 μm, morepreferably less than about 50 μm, and most preferably less than about 30μm, 25 μm or even less than about 20 μm or 15 μm. In certainembodiments, the diameter of the internal channel expands to a maximumof about 100 μm, preferably about 75 μm, more preferably about 50 μm. Inone embodiment, illustrated in FIG. 2, the spotting capillary has anaperture diameter of about 30 μm or less and a maximum internal diameterof about 75 μm or less. The capillary holds about 0.2 μL or less,preferably about 0.1 μL or less.

[0054] The spotting capillary outer diameter determines the minimuminter-pin (inter-capillary) spacing, and the inter-pin spacingdetermines the minimum spacing of the reservoir(s) from which the printhead can load reagents. Preferred spotting pins have an outside diameterof about 1 mm or less, more preferably of about 0.7 mm or less, and mostpreferably of about 0.4 mm or less. The 0.4 mm spotting capillaries canbe mounted very close together and, with such close spacing, the printhead can load reagents from standard 96 well, 384, well, 864 well, and1536 well microtiter plates. In preferred embodiments, the center tocenter spacing of the spotting capillaries is about 10 mm or less,preferably about 5 mm or less, more preferably about 3 mm or less, andmost preferably about 2 mm or even 1 mm or less. Such close spottingcapillary spacing can be achieved, e.g. using the print head designsillustrated herein, allowing the use of even higher density samplereservoirs.

[0055] The support(s) for the spotting capillaries can take any of anumber of a number of forms. For example, in one embodiment, the supportcan comprise a number of channels drilled, etched, or cast in a singlemetal or plastic piece. The channels then act as guides for the spottingcapillaries. Alternatively, the support can be fabricated as by joininga collection of tubes e.g. metal tubes. The tubes can be glued or weldedtogether to form a single support structure, each of the tubes acting asa channel for housing a spotting capillary.

[0056] In a particularly preferred embodiment, as illustrated in FIG.1A, the spotting capillaries are supported and positioned by a series ofguide plates 14, and optionally, by a guide cylinder 16. To preventbreakage of the spotting capillaries e.g., do to the repetitivecontacting with the printing substrate, the spotting capillaries arecapable of sliding through the guide plates, e.g. when they contact thespotting substrate. The spotting capillaries are then returned to their“extended” position by a spring 18.

[0057] While FIG. 1A is illustrated with a coiled spring, it will beappreciated that any of a variety of springs can be used. These include,but are not limited to deformable elastic masses, deformable elasticmembranes, “rubber bands”, air pressure, and the like.

[0058] The extended position of the spotting capillaries is limited by adetent 20. The detent can take any convenient form. For example, thedetent could comprise a set screw (preferably plastic so as not todamage the spotting capillary), a drop of epoxy or other resin, and thelike. In one particularly preferred embodiment, the detent is a diskattached to the spotting capillary. In one embodiment the detent stopsup against the pin guide plate The “downward” extent of the spottingcapillary can be determined by the position of attachment of the detentto the spotting capillary. Alternatively, the guide plate can furthercomprise an adjustment means (e.g. a set screw, a shim, etc.) for eachspotting capillary that can be used to adjust the downward travel foreach spotting capillary.

[0059] In certain embodiments, the spring typically rests against aresisting surface, e.g. a spring capture plate 22.

[0060] It was also a discovery of this invention that spottingcapillaries, particularly when fabricated of glass, quartz, otherminerals, or ceramic or porcelain, show a dramatically improvedlifetime, when the outer edges of the spotting tip of the spottingcapillary, are not flush with the spotting face. Thus, in preferredembodiments, the outer edge of the spotting tip is beveled (see, e.g.,30 in FIG. 2).

[0061] The print head typically comprises a plurality of spottingcapillaries. Preferred print heads comprise at least two spottingcapillaries, more preferably at least 4 spotting capillaries, still morepreferably at least 8 or at least 16 spotting capillaries, and mostpreferably at least 32, 64, 128, or 256 spotting capillaries. Dependingon the application, a print head can be configured to use fewer than themaximum number of available spotting capillaries. FIG. 1B illustrates aprint head capable of mounting 64 pins on 3 mm centers for 864 wellmicrotiter plates. In this instance, 16 pins are in use.

[0062] The print heads of this invention can be fabricated usingstandard machining and glass handling techniques well known to those ofskill in the art. The spotting capillaries are preferably fabricated bycasting or by pulling a quartz or glass microcapillary tube using acommercially available microcapillary puller (e.g. Sutter InstrumentP-2000 Capillary Puller). In particularly preferred embodiments, themicrocapillary tip is then beveled using a glass grinder. Such spottingcapillaries can be made to order by commercial production houses.

[0063] II. Magnetic Control of Printing Pins/Capillaries.

[0064] In certain embodiments, the printing capillary position can becontrolled by magnetic methods. Both passive and magnetic controlmethods are contemplated by this invention.

[0065] A) Passive Magnetic Control of Printing Pins/Capillaries.

[0066] A passive magnetic control system is schematically illustrated inFIG. 13. This figure illustrates a portion of a print head comprising aprinting capillary 12 and guide plates 14 a and 14 b. The capillarieshave affixed thereto a magnetic material 202. Similarly, guide plate 14a can be a magnetic material. One or both of 202 and 14 a is magneticand a “passive” magnetic attraction “M” pulls the capillary 12downwards. The downward travel of the capillary is stopped by optionalspacer 200.

[0067] The open arrow indicates the direction of motion of the printhead during a printing operation. When the print head is loward, theprinting capillaries contact the array substrate. The are then no longerheld doun by the magnetic force “M”. When the print head is raised, themagnetic attraction between 200 and 14 a pulls the pins/capillaries backto the reference position. This system makes is easy to change printingpins. They can be changed just by pulling them out of the top of theprint head.

[0068] B) Active Magnetic Control of Printing Pins/Capillaries.

[0069] An active magnetic control system is schematically illustrated inFIG. 14. In this embodiment, the printing pins are contacted with thearray substrate using electromagnetic means. The system offers twoadvantages. First, the printing pins move, while the print head remainsstationary, so printing speed is increased. Second, the printing pinscan readily be changed, simply by lifting them out of the top of theprint head.

[0070] In this embodiment, guide plate 14 a is an electromagnet. Eachprinting pin/capillary has affixed thereto a material 202 that isattracted by a magnetic field (e.g. a ferrous material, a magnet, etc.).A spring or elastic material 204 lifts the pins when the electromagnetis not activated. When the electromagnet 14 a is activated, the pins aredrawn downward to contact the array substrate thereby depositing arrayfeatures.

[0071] The system is simple to build since only one electromagnet isrequired regardless of the number of pins/capillaries.

[0072] III. Platens for Array Substrate and/or Print Head Positioning.

[0073] To reliably print an array at high feature density (spots/cm²) itis desirable to reliably and consistently position the spottingcapillaries on the microarray substrate. The more precisely andconsistently the print head can be positioned relative to the microarraysubstrate(s), the more possible it becomes to print arrays at a higherfeature density.

[0074] Printing a larger number of arrays at a time, however, requires alarger array substrate support (platen). The larger the platen, the moredifficult it becomes to reliably and consistently position it relativeto print head. One approach to solve this problem is illustrated in FIG.3. The platen illustrated on the left utilizes two guide rails 42 tominimize torque and yaw and hysteresis of the platen introduced by theactuator which moves the platen, e.g. in the ±Y direction. To accuratelyprint arrays, it is desirable to accurately position the platen totolerances better than 50 μm. Such precise positioning requiresextremely good bearing alignment for the four bearings and the guiderails must be extremely parallel. The rails and bearings must stayaligned throughout the printing operation. One of skill in the art willappreciate that such a device will tend to jam and/or introducepositioning imprecision as the actuator drives the platen through jammedpositions if the alignment is not adequate. Leaving “play” in thebearings to allow motion results in positional imprecision. In stillother standard types of arrayers the guide rails, e.g. as illustrated inthe embodiment on the left in FIG. 3 are incorporated into the actuator.These rails are closely spaced and permit slight random yaw motions ofthe platen. For large platens, even this slight yaw results in apositional uncertainty that increases as one moves away from the centerof the platen and decreases array element accuracy and therefore arrayelement density.

[0075] Such difficulties are solved with the platens of this invention.One embodiment of a platen 40 of this invention is illustrated by theplaten on the right in FIG. 3, and in FIG. 4. In certain embodiments,the plattens of this invention utilizes a single guide rail 42 toconstrain the position of the support surface 44 that bears the arraysubstrates 56. The guide rail is typically coupled to the platen throughone or more bearings that permit motion of the platen in one direction(along one axis), but restrict motion in other directions. In certainpreferred embodiments, the guide rail/platten configuration is designedto control the motion each degree of freedom of the spotter (e.g. theplatten and/or print head) with the minimal number of restraints thatare required by Euclidian geometry. Thus, for example, two pointsdetermine a line, three points determine a plane etc. To limit degreesof freedom around the guide rail, two coupling points (e.g. bearings)are used to constrain the direction of motion (e.g. in a line), and acoupling point is used at a location off of the line to limit thedegrees of freedom of the platten, a plane. The attachment to this thirdpoint provides some flexibility in certain degrees of freedom etc. sincethe guide rail may not be accurately straight. In this example, it isnot desired to place three bearings on the guide rail since that is morethan required and if the rail is slightly curved the bearings will bind.Similarly one bearing on the rail is generally insufficient, becausethat will allow the platten to rotate.

[0076] Illustrative embodiments, of these principles are provided inFIG. 3, and in FIG. 4. As illustrated, the platen utilizes a singleguide rail 42 to constrain the position of the support surface 44 thatbears the array substrates 56. The support surface communicates with aguide rail 42 through two bearings 46, the minimal number required todefine linear motion. The support surface is coupled to an actuator 48through a flexible coupling 50.

[0077] The bearing(s) 46 and the guide rail 42 prevent the platten(e.g., the support surface) from yawing in response to a force createdby the actuator. However, because there is only a single rail, there areno difficult alignment problems. Straightness of the guide rail is alsonot critical because any rail deformation will be constant andreproducible, i.e., repeated positioning of the array substrates will beconsistent, and the bearings will not bind due to possible curvature inthe rail. An encoder 58 accurately encodes the location of the platen.The encoder can be external to the actuator as shown in the drawing, orit can be built into the actuator as is common in commercially availableactuators.

[0078] The support surface is coupled to the actuator through a flexiblecoupling 50 that is rigid in the direction of travel (±Y direction inFIG. 3), but compliant in all other directions. This permits theactuator to accurately position the platen (e.g. in the Y direction asshown), while not jamming or binding in other directions.

[0079] The distance between the two bearings on the guide raildetermines how much yaw motion will be permitted in the platten. If thebearings allow a certain lateral motion, that will be translated into ayaw of the platten. The positioning precision is related to the lengthand width of the slide platten in relation to the distance between thetwo bearings on the guide rail. It is desirable to provide very highreproducibility in the positioning of the plattens, but the absoluteaccuracy is not so critical. Thus if the guide rail is curved a bit, theplatten will yaw as it moves, but that motion will be very reproducibleso that the next array spot is printed, it is properly placed relativeto the previous spots, although the entire array on one substrate willbe positioned slightly differently than on a substrate at a differentlocation on the platen. Thus the system is designed to give extremelygood performance on the relative positions of spots in an array.

[0080] The embodiment illustrated in FIG. 4 allows slight motion in theX direction, and yaw, roll and pitch compliance. The system is stiff inthe Y direction. One thing to note is that with the flexing, especiallypitch, the locations on the platen will vary compared to the encoder,when the encoder is on the motor (actuator) carriage. As long as thesevariations are reproducible, then the positioning is reliable.

[0081] The flexible coupling 50 illustrated in FIG. 4 is a flexiblesheet (e.g., sheet metal. This flexure gives freedom in yaw, roll and X,and is stiff in Y, the direction of platen travel. If pitch freedom isdesired, it is possible to introduce additional flexible couplings 54.Alternatively, a bearing can be used. The two flexures 54 will be stiffin the Y direction as long as the platen does not lift up underacceleration, and will be stiff in yaw so that all of the yaw compliancewill be taken care of in the vertical sheet.

[0082] In certain embodiments, a pivot 52 is provided. In variousembodiments, the pivot 52 includes, but is not limited to two pointsacross the width (into the drawing in the side view), a cylinder etc. Inthis design, pitch motion will require the upper mounting plate to slideacross the pivot, so this is preferably lubricated and/or fabricated oflow-friction materials, etc.

[0083]FIG. 5 illustrates one embodiment of an array substrate platen inan exploded view.

[0084]FIG. 6 illustrates one embodiment of a platen used to move theprint head in a microarray printer of this invention in a ±X direction(normal to the direction of motion of the array substrate platen). Inthis embodiment, the platen (support surface) is oriented vertically. Amicroarray print head 10 is attached to the support surface 44. In theembodiment illustrated in FIG. 5, the actuator 48 is a motor (e.g. alinear stepping motor). The actuator 48 is coupled to the supportsurface through a flexible coupling 50. The couplings are disposed suchthat deflection of the support surface is constrained (stiff) in the Xdirection (the direction of motion), and stiff in the Y direction, butcompliant in yaw. The coupling 50 a flexes to relieve Z, yaw, and rollof the platten. The platen rides along a truss 58 that bears a guiderail 42. In a preferred embodiment, the support surface 44 istrapezoidal in shape with the wide side of the trapezoid disposed alongthe guide rail. This permits the rail bearings to be widely separatedand thereby minimize rotation of the support surface. Use of thetrapezoidal shape minimizes support surface mass permitting more rapidacceleration and deceleration. The print head 10 is mounted on a Z stage60 that controls vertical movement of the print head.

[0085] Because the moment arm between the flexible motor coupling 50 andthe print head is large compared to the moment arm between the printhead and the guide rail 42, rotations or deflections at the motorcoupling have minimal effect on the position of the print head.

[0086] Using the teachings provided herein, one of skill would recognizenumerous embodiments for the flexible couplings used in the platens ofthis invention. In certain embodiments, as indicated above, the flexiblecouplings comprise flexible sheets (e.g. sheets of metal, plastic, orother flexible material). The sheets are selected of materials that arestiff in tension, but capable of bending in other directions. Otherflexible couplings include, but are not limited to ball bearings, rodbearings, pin bearings, and the like.

[0087] A wide range of encoders can be used to encode the position ofthe platen/support surface. Encoders are well known to those of skill inthe art and include, but are not limited to optical encoders, mechanicalencoders, magnetic encoders, and electronic encoders. Various electronicencoders include, but are not limited to encoders that convert thechange in resistance of a potentiometer or the change in capacitance ofa capacitor into a movement or position. Optical encoders include, butare not limited to encoders that convert an optical signal, e.g. a barcode, an interferometric measurement, etc. into a movement or position.Similarly, magnetic encoders include encoders that a change in magneticflux or field into a movement or a position. Suitable encoders (e.g.with a positional accuracy greater than about 50 μm, preferably with apositional accuracy greater than about 25 μm, more preferably with apositional accuracy greater than about 10 μm, and most preferably with apositional accuracy greater than about 5 μm, greater than about 2 μm, orgreater than about 1 μm are commercially available.

[0088] The actuator can be any device or means capable applying a forceto the platens of this invention and driving them in a plus or minusdirection along the guide rail. Suitable actuators include, but are notlimited to stepping motors, linear induction motors, pneumaticactuators, solenoids, piezo-electric actuators, lead screws, and thelike. Suitable actuators, and associated motion control products arecommercially available from a wide variety of companies (see, e.g.,Biorobotics (U.K), Parker Daedal, Irwin, Pa., and the like).

[0089] It is noted that in certain preferred embodiments, of the presentinvention, the slide support platen, with an encoder precision of about2 μm achieves a spot (array element) precision of about 10 to about 20μm at any array substrate location on the support surface. Particularlypreferred platens achieve a spot (array element) precision of betterthan about 5 μm, more preferably better than about 2 μm, and mostpreferably better than about 1 μm, or 500 nm, or 200 nm at any arraysubstrate location on the support surface.

[0090] Similarly, the print head positioning platen achieves a precisionof about ±10 μm or less, more preferably about ±5 μm or less, and mostpreferably about ±3 μm or less over the entire slide (array substrate)support surface.

[0091] IV. Positive and Negative Pressure Control for Sample Loading andDispensing.

[0092] A) Pressure Control Sytems.

[0093] In a particularly preferred embodiment, the microarray printingdevices of this invention utilize positive pressure and negativepressure (vacuum) to control sample loading and dispensing. Each“active” spotting capillary 12 is in fluid communication, e.g. viacapillary tubing 70 with a manifold 64 (see, e.g., FIGS. 6, 7, 8, and 9)that permits the application of pressure or vacuum to the spottingcapillaries.

[0094] A preferred plumbing scheme is illustrated in FIG. 7. Inpreferred embodiments, the gas flow (e.g. air, nitrogen, argon, etc.)flows through the manifold always in the same direction for alloperations. This assures that any liquid drops that may be left in thetubing always will be forced to move toward the waste bottle and willnot be blown back into the manifold. If droplets do get into themanifold they may block the supply of printing pressure to one or morepins, thus reducing the reliability of the printing.

[0095] In general, the plumbing system comprises a pressure source 78,and a vacuum source 80. The pressure and vacuum sources are in fluidcommunication with a manifold 64, e.g. via tubing 84. The manifold isalso in fluid communication with the spotting capillaries so thatpressure or vacuum applied to the manifold is delivered to the channel(bore) in the capillaries. In preferred embodiments, there is a wastereceptacle disposed between the low vacuum source 80 and the manifold74. In certain embodiments, vacuum source 80 supplies various levels ofvacuum.—a “high” vacuum for cleaning and for starting the filling of thepins with a vacuum pulse, and a “low” vacuum that is sometimes used tohelp capillary action complete the filling of the pins. This low vacuumis typically low enough so that the printing liquid is not pulled pastthe top of the printing pins and into the flexible capillary tubing.

[0096] In preferred embodiments, the system is designed so that thetubing from the waste bottle to the manifold preferably slopes downwardto facilitate liquid flow, and we strive to minimize the volume of thetubing and waste receptacle so that the pressure changes are transmittedto the manifold quickly. Thus, while the tubing communicating thepressure and vacuum to the manifold and communicating the manifold tothe waste receptacle can be essentially any convenient tubing (as longas it is resistant to the reagents employed), in preferred embodiments,the tubing is a low void volume tubing (e.g. a fine bore capillarytubing), but the diameter is not so small as to introduce too much flowresistance. Such tubings are well known to those of skill in the art.

[0097] In one preferred embodiment the pressure source 78 can apply twopressures, a blowout pressure (e.g. 15 PSI), and a positive pressureused during printing (e.g. from about 0.1 to 2, preferably from about0.1 to 1, more preferably from about 0.1 to about 0.5, and mostpreferably about 0.3 inches of water). Similarly, in preferredembodiments, the vacuum source 80, can apply two pressures, a “highvacuum” for cleaning, and for starting filling of the tubes using ashort burst of vacuum, and a “low vacuum” to assist capillary action infilling the printing pins. The waste receptacle is preferably a lowvolume waste receptacle (e.g. a 200 ml waste bottle).

[0098] One suitable manifold is illustrated in FIG. 8. This manifoldcomprises a common channel with an inlet port (manifold inlet) 66 and anoutlet port (manifold outlet) 68. In fluid communication with themanifold are a number of capillary connectors 76 that each with aninternal (manifold) capillary port 72, and an external capillary port68. Each capillary connector is disposed to receive a connection (e.g. atubing connection) to provide a fluid communication to a spottingcapillary 12. The internal capillary port 72 is disposed inwards intothe manifold so that the capillary port is not flush with the internalwall of the manifold. This prevents droplets from accumulating on theinternal capillary port 72 which could interfere with reliable loadingor delivery of samples.

[0099] The manifold can be made of any of a variety of materials and cantake a number of different shapes. Preferred shapes however, permit therapid distribution of pressure, permit the unidirectional flow of gasand waste, and permit the disposition of the internal ports 72 away fromthe internal surface of the manifold. Useful materials, include variousplastics, glass, quartz, ceramic, and metals. In one preferredembodiment, the manifold is fabricated from stainless steel.

[0100] A plumbed print head is illustrated in FIG. 9. This figureillustrates the print head 10 comprising a plurality of spottingcapillaries 12 (four visible in the figure). The spotting capillariesare in fluid communication with the manifold 64 via flexible capillarytubing 70. A pressure line 84 a can be seen at the upper right and awaste/vacuum line 84 b can be seen at the upper left.

[0101] Another pressure/vacuum system is illustrated in FIGS. 10, 11 and12. This pressure/vacuum system for managing the print head permitsoperation in a larger format printer where tubing between the print headand pressure and vacuum sources is sufficiently long so that the volumeand flow impedance of the tubing has a significant impact on printeroperation. The basic operating concept is to assure that flow of gas orliquid in the manifold is always towards the waste bottle as shown bythe arrow in FIG. 10. This prevents liquid that may be in present theline going from the manifold to the waste bottle from flowing back intothe manifold and blocking the tubes that supply pressure and vacuum tothe printing pins. When large amounts of liquid are present in themanifold, for example during a wash cycle, sufficient air flow ismaintained in the manifold to rapidly sweep the fluid into the wastebottle. The controls for the pressure side of the system are illustratedin FIG. 11, and those for the vacuum side are illustrated in FIG. 12.

[0102] As shown in FIG. 11, A pressure source 98 (e.g. N2 tank) isconnected through a printhead supply valve V1 100 to a medium (blowout)pressure regulator R2 102 (e.g. ˜20 psi) and to a very low (printing)pressure (e.g., ˜0-0.5 in H₂O) regulator R3 104. The regulators areoptionally connected to a blowout pressure readout 106, and/or to a verylow pressure readout 108. The very low pressure side is connectedthrough a very low (printing) pressure valve V3 110 to a line 112leading to the high pressure side of the manifold 64. The mediumpressure side is connected through V3 medium (blowout) pressure valve V2114 to a line 90 leading to the high pressure side of the manifold 64.The low pressure side is also vented to a vent through vent valve V0 116to a vent 118.

[0103] As shown in FIG. 12 vacuum source 80 is connected to a coarsevacuum regulator R4 132 maintaining a “vacuum” of about 20 in Hg, and toa medium vacuum regulator R5 134 maintaining a “vacuum” of about 15 inHg. The two regulators can, optionally, be connected to “coarse vacuum”and medium vacuum readouts 136 and 138, respectively. The line from thecoarse side vacuum regulator connects via a coarse (wash) vacuum valveV4 140 to a waste receptacle 82 which is also connected via a vacuumline 146 to the manifold 64 as shown. The line from the coarse sidevacuum regulator connects via a vacum valve V5 142 to the wastereceptacle 82 and via a vacuum vent valve VV 144 to a vent 72 asillustrated.

[0104] Two features contribute to maintaining unidirectional flow andrapidly clearing liquid from the manifold. Liquid clearing is assured bythe vent line 120 containing connecting to the vent 118 and containing aneedle valve 122, which is controlled by the “constrictor valve” 116 inFIG. 11. This has particular use when washing the print head. At sometimes during the washing the tips of the printing pins are immersed inliquid and a vacuum is applied. This draws liquid into the pins and upinto the manifold. Opening constrictor valve 116, allows a small amountof air to enter the pressure side of the system, and sweeps the liquidout of the manifold and to the waste bottle. The amount of flow isdetermined by adjusting the needle valve.

[0105] Unidirectional flow is accomplished always applying vacuum to oneside of the manifold, and pressure to the other, and by providingatmospheric vents on both the pressure and vacuum sides of the system.The vents are controlled by valves V0 116 and VV 144 respectively. Incombination with valve CV 116, this permits rapidly returning the systemto atmospheric pressure without having a period of flow in reversedirection.

[0106] For example, during a wash cycle where vacuum is applied, all ofthe tubing in the system including that on the high pressure side of themanifold, is below atmospheric pressure. If the only atmospheric ventwere on the vacuum side of the system, opening that vent would result inflow from the low pressure side toward the manifold as the tubing filledwith air. Conversely, when the system is pressurized in order to blowout any liquid that may be in the printing pins, the tubing and wastebottle are at elevated pressure. If the only atmospheric vent were onthe high pressure side of the system, then the gas in the waste bottlewould flow towards the manifold when this vent was opened. Both of thesereverse flows may be large enough to transport any liquid that may be inthe tubing connecting the manifold to the waste bottle back towards themanifold, perhaps causing some to enter the manifold. The flow reversalscan either be entirely eliminated or reduced to acceptable levels byusing vents on both the pressure and vacuum sides of the system. Openingboth vent valves, V0 116 and VV 144, simultaneously permits returningthe system to atmospheric pressure without substantial flow reversal.The dual vents V0 and VV also provide the advantage of returning thesystem to atmospheric pressure rapidly since VV can be physically closeto the waste bottle, reducing the flow impedance between it and thevent.

[0107] B) Representative Print Cycle.

[0108] Valve V1 100 is typically open continually during printing. Atypical print operation begins by cleaning the printing pins. The tipsof the pins are dipped into a sonication bath containing a dilutesolution (approximately 1 part in 10,000 in distilled water) of glasscleaning solution, (Micro 90). Pressure may be applied to expel thecontents of the pins by opening valve V2 114 or V3 110. Subsequently,vacuum is applied to the manifold by opening valve V4. Valve CV is alsoopened to provide air flow through the manifold. The vacuum drawscleaning fluid into the printing pins and up to the manifold, where theair flow sweeps it rapidly to the waste bottle. The print head is dippedinto the cleaning fluid several times with the vacuum applied so thatsome air enters the printing pins when they are raised above the fluid.Each dip cycle last approximately 1 sec. Alternating regions of air andliquid travel up the printing pins and their associated tubing to themanifold. The interspersed regions of liquid and air provide more rapidremoval of residual printing solutions from the printing pins than wouldbe obtained if the tips of the pins were just dipped into the washsolution and remained continuously immersed.

[0109] The print head is then moved over a second sonication bath thatcontains distilled water and the tips of the pins are periodicallyimmersed several times with valves V4 140 and CV 116 open. Each dipcycle last approximately 1 sec. This draws water through the pins toremove the cleaning solution. The print head is then moved to a dryingstation where warm air is blown over the printing pins for approximately5-10 seconds. During this time, pressure may be applied for a period toblow liquid out of the pins by opening V2 114 and closing the othervalves. The remainder of the drying time vacuum is applied by opening V4140, and CV 116 is opened to allow airflow to dry the manifold.

[0110] After the drying period V4 and CV are closed and atmosphericvents VV 144 and V0 116 are opened to bring the system to ambientpressure. The print head is then positioned over the microtiter plateand the tips of the printing pins are immersed in printing solutionsfrom the desired position in the plate. VV 144 and V0 116 are closed. Avacuum pulse is applied by opening V5 142 in order to begin the loadingof printing solutions into the pins. After ˜0.1-. 1.0 sec V5 is closedand the atmospheric vents VV 144 and V0 116 are opened to return thesystem to ambient pressure. Loading of the printing solutions continuesfor ˜1-2 sec using capillary action. The printing pins are then liftedout of the printing solutions and VV and V0 closed.

[0111] The print head is then moved to its printing position and valveV3 110 is opened to apply a low, constant, pressure of 0.0-0.5 inches ofwater to the printing pins. This pressure assures that the printingsolutions remain at the tips of the printing pins so that the tipsremain wet with printing solution. Varying the pressure provides somecontrol over the amount of liquid that is deposited when the printingpins contact the array substrates. The pressure is kept low enough sothat the printing solutions are not ejected from the pins. The tips ofthe pins are contacted with the array substrates at all of the locationsspecified by the operator. After completion of the printing with thecurrent load of printing solutions, the cycle starts again with theprint head being moved over the first sonicator bath, the tips loweredinto it, and the unused printing solutions expelled.

[0112] V. Preparation of a Microarray.

[0113] The microarray printer of this invention can be used to printmicroarrays comprising essentially any molecules that can be suspended,dissolved, or otherwise placed in a solution. Preferred microarraysinclude, but are not limited to microarrays of biomolecules (e.g.sugars, carbohydrates, nucleic acid, proteins, and the like).Particularly preferred microarrays include nucleic acid and/or proteinarrays. Methods of preparing and/or purifying biomolecules are wellknown to those of skill in the art (see, e.g., Berger and Kimmel (1989)Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif.; Sambrook et al. (1989)Molecular Cloning-A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY; Ausubel et al. (1994)Current Protocols in Molecular Biology, Current Protocols, a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc., (1994 Supplement) (Ausubel); U.S. Pat. No. 5,017,478; andEuropean Patent No. 0,246,864.).

[0114] The materials that are to be printed (e.g. proteins, nucleicacids, etc.) are typically formulated in “printing solutions”. Solutionsfor microarray printing are well known to those of skill in the art(see, e.g., U.S. Pat. Nos. 6,101,946, 5,958,342; and MacBeath andSchreiber (2000) Science 289: 1760-1763; Mark Schena (Ed.) (1999) Genes,Genomes and Chips. In DNA Microarrays: A Practical Approach, OxfordUniversity Press, Oxford).

[0115] The microarrays can be fabricated on any of a wide variety ofsubstrates well known to those of skill in the art. Such substratesinclude, but are not limited to glass, plastic, quartz and otherminerals, metal, ceramic, porcelain, metal covered (e.g. sputtered)glass, and the like. A number of substrates, often derivatized tofacilitate microarray printing are commercially available (see, e.g.,silane slides from Sigma Chemical Co., the SuperClean™, SuperAmide™, andSuperAldehyde™ substrates from Telechem, International Inc., etc.).

[0116] In operation, the printing pins (spotting capillaries) areinitially washed. In a preferred embodiment, this involves moving theprint head over a wash bath and applying pressure (e.g., about 15 PSI)to the manifold to blow out any liquid remaining in the spottingcapillaries. The spotting capillaries are dipped in and out of acleaning solution (e.g. 0.001% Micro-90, Cole Palmer Inc.), in asonicating bath at about 0.75 sec intervals for about 3 cycles whilepressurized.

[0117] The device is switched from pressure to house (“high”) vacuum andthe spotting capillaries are dipped into the sonicated cleaning solutionfor 3 more cycles while drawing cleaning solution into the spottingcapillaries. The spotting capillaries are then moved to a sonicatingrinse bath that contains pure (e.g. double distilled) water.

[0118] The spotting capillaries are dipped in and out of water for about3 cycles of about 0.75 sec each, with vacuum applied. This allows theformation of interspersed air bubbles and water in the tubing, assuringthat the cleaning solution is more effectively removed from the walls ofthe tubes. Finally the spotting capillaries are dried by sucking airthrough them using house vacuum, blowing hot air over the spottingcapillaries for about 4 sec, opening a vent to atmosphere and continuingvacuum for about 2 sec in order to clear liquid from the manifold andwaste tubing (see FIG. 7).

[0119] The spotting capillaries are then filled by dipping the spottingcapillaries into reagent reservoir(s) (e.g. a microtiter plate)containing the printing solution(s). Full house vacuum is applied to themanifold for about 0.15 sec in order to assure that the solutions enterthe tips of the pins. The manifold is then vented to atmosphericpressure and the spotting capillaries are allowed to sit in the printingsolutions for about an additional 0.75 sec so that capillary actionfills the spotting capillaries. In one preferred embodiment, thespotting capillaries each contain approximately 0.2 ml when full. Thespotting capillaries do not fill beyond their tops due to capillaryaction—the solutions do not enter the flexible tubing that connects thepins to the manifold. In some cases a slight vacuum of ˜0.2 inches ofwater is used to assist the filling. This is adjusted to be low enoughso that the no liquid is drawn beyond the top of the pins. The entirewash, dry and fill functions take about 25 seconds.

[0120] To print an array feature or features, the print head is movedover the first printing substrate and lowered to make contact, andraised. In one preferred embodiment, in the upper position the tips ofthe printing pins are about 0.5 to 1.0 mm above the array substrate, andwhen printing the print head is lowered so that the pins would moveabout 0.2 mm below the substrate surface if no array substrate werepresent. When a substrate is present the spotting capillary tips contactit and the spring mounts allow the spotting capillaries to stop movingwhile the print head body continues its motion toward the substrate. Ina preferred embodiment, the total time for the print head to move down,contact the slide and return to the upper position is about 0.05 to 0.2sec. During the printing operation a constant pressure of 0.1 to 0.4inches of water is applied to the manifold to keep the printingsolutions at the tips of the spotting capillaries. This assures that thespotting capillaries are wet with the printing solutions so that liquidwill be transferred to the substrate on contact. The pressure does noteject the printing solution. If this is not done, the solutions can pullaway from the tips and the printing will stop. The small diameter of thetubing and the printing pins provides enough flow resistance to air sothat the manifold pressure is maintained even if one or more of the pinsdoes not contain printing solution. The cycle is repeated to printadditional features or other array substrates.

[0121] The amount of printing solution that is deposited on thesubstrate depends on the interaction between the substrate and theprinting solution, and the diameter of the tip of the printing pin. Whenprinting salt solutions on glass, each fill of the printing pin (0.2 ml)can make at least 10,000 spots. When printing 20% DMSO solutions withDNA, the same load can print at least 2000 spots. This is many morespots than are possible with other printing systems. Thus the systemdescribed above can deposit less than 100 pL per spot in typicalprinting.

[0122] Use of the novel spotting capillaries and/or print heads of thisinvention provides extremely efficient reagent usage. In certainembodiments, the printer can print at least about 500 spots (features)per 0.2 μL load, more preferably at least about 1000 spots (features)per 0.2 μL load, most preferably at least about 1500 spots (features)per 0.2 μL load, at least about 2000 spots (features) per 0.2 μL load,at least about 5,000 spots (features) per 0.2 μL load, or at lest about10,000 spots (features) per 0.2 μL load. Because the printing capacitiesare so high, the print head typically does not need to refill during aprint run. This greatly decreases the duration of a print run when alarge number of arrays are made at one time.

[0123] VI. Microarray Printing Device.

[0124] The various elements described above, e.g. spotting capillaries,print head design, array support platen, print head support platen,vacuum and pressure system, manifold, sample loading unloadingprotocols, and the like can be incorporated individually, or incombination, into preexisting microarray printers or they can beassembled into a microarray printer built de novo.

[0125] Methods of designing and building microarray printing devices aregenerally known to those of skill in the art (see, e.g., U.S. Pat. Nos.6,110,426, and 5,807,522, and publications of the Brown Laboratory atStanford University (e.g., The McGuide. Version 2.0, available on theinternet at http://cmgm.stanford.edu/pbrown/mguide/index.html, and fromCold Spring Harbor Laboratories).

[0126] In general, microarray printers of this invention will, inpreferred embodiments, include a base adapted to hold reservoir(s) ofprinting solutions, a platen for supporting and positioning microarraysubstrates, and a platen for supporting and positioning a print head.The microarray printer will include actuators (e.g. motors) fordriving/positioning the various platens and for vertically positioningthe print head. The microarray printer, will typically includeassociated electronics to read encoded platen positions and/or to drivethe various actuators to position the array substrates and print head.Typically such electronics will include a computer controller. Themicroarray printer can additionally comprise vacuum and pressure lines,reagent reservoirs, waste receptacles, cleaning baths and the like asdescribed herein.

[0127] In a particularly preferred embodiment, the microarray printerwill include the print head platen, the array substrate platen, a printhead comprising spotting capillaries as described herein. The microarrayprinter will also preferably also include pressure and vacuum sources asdescribed herein. While it is preferred that the microarray printercomprise all of the elements described herein, it is not required thatall such elements be present. Thus, in certain embodiments, themicroarray printer comprises one, two, or only a few of such elements.Thus, for example certain microarray printers may only comprise a printhead according to this invention and/or the array substrate platen,and/or the print head support platen, and so forth.

[0128] VII. Microarrays.

[0129] It is believed that the microarray printers of this inventionpermit the production of spotted microarrays with an accuracy,consistency, and array feature density previously unavailable. Thus, incertain embodiments, this invention provides high-density microarrayscomprising a plurality of molecules, preferably biomolecules where thearray comprises at least about 1,000 features (spots), preferably atleast about 10,000 features (spots), more preferably at least about40,000 features (spots), and most preferably at least about 100,000features (spots), or at least about 1,000,000 features (spots). Inparticularly preferred embodiments, the features are present at anaverage center-to-center spacing of about 130 μm or less, preferablyabout 100 μm or less, more preferably about 80 μm or less, and mostpreferably about 65, 50, or 40 μm or less.

[0130] In certain embodiments, the microarray is a protein and/ornucleic acid microarray. In nucleic acid arrays unlike chemicallysynthesized arrays, the printed arrays of this invention are not limitedby the size of nucleic acid. Large nucleic acids can be printed aseasily as small nucleic acids (e.g. oligonucleotides less than 20-30mer). Indeed, there is no size limit on the printed nucleic acids andthe particular nucleic acid sizes depends on the intended use of thearray. Arrays printed according to the methods of this inventiontypically have a fragment size ranging from about 100 to about 1000bases (e.g. a mixture of PCR fragments). Thus, frequently nucleic 100nucleotides or longer are printed. Fragment sizes ranging from about1000 bases to about 10,000 bases or from about 10,000 bases to about100,000 bases or larger can be readily accommodated.

[0131] Preferred arrays of this invention have a feature (spot) densitygreater than about 5,000, 10,000, or 20,000 features/cm², preferablygreater than about 30,000 features/cm², more preferably greater thanabout 40,000 features/cm², and most preferably greater than aboutgreater than about 50,000 or 60,000 features/cm².

[0132] Because the the reagents are typically simply spotted, inpreferred embodiments, the molecule(s) comprising the array features aresimply adsorbed to said substrate. However, in certain embodiments thereagents and/or the array substrate can be derivatized so that themolecules comprising the features (spots) covalently couple to thesubstrate. Methods of so derivatizing macromolecules are well known tothose of skill in the art. Thus, for example, the reagents can bederivatized with a sulfhydryl group (—SH) which will covalently coupleto a gold surface (e.g. gold coated glass).

[0133] VIII. Kits.

[0134] In still another embodiment, this invention provides kitscomprising one or more containers containing the arrays described above.In certain embodiments, the arrays will comprise features representingnucleic acids from every chromosome in a subject organism (e.g. ahuman). In certain embodiments, the arrays will comprise featuresrepresenting nucleic acids from every known expressed sequence tag (EST)for a given organism, or tissue, or whose expression is associated witha particular physiological state (e.g. a particular pathology).

[0135] These array constituents are merely illustrative. Numerous otherarrays components will be recognized by one of ordinary skill in theart.

[0136] In certain embodiment, the kits can, optionally, additionallycontain one or more of the following: detectable labels, hybridizationreagents, software, buffers, and the like.

[0137] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the purview of this application andthe scope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A spotting capillary for use in a microarrayprint head, said spotting capillary comprising: a capillary comprising amicrocapillary comprising a tapered tip.
 2. The spotting capillary ofclaim 1, wherein said spotting capillary is a microcapillary.
 3. Thespotting capillary of claim 2, wherein said tapered tip is ground. 4.The spotting capillary of claim 1, wherein said spotting capillary isfabricated by heating and pulling a quartz or glass microcapillary tubeand then beveling the tip using a glass grinder.
 5. The spottingcapillary of claim 2, wherein said spotting capillary has maximum loadvolume of about 0.5 μL.
 6. The spotting capillary of claim 2, whereinsaid spotting capillary has a minimum load volume of about 0.05 μL. 7.The spotting capillary of claim 1, wherein said spotting capillary has aload volume of about 0.2 μL.
 8. The spotting capillary of claim 1,wherein said spotting capillary is fabricated from a material selectedfrom the group consisting of glass, a mineral, ceramic, and porcelain.9. The spotting capillary of claim 8, wherein said spotting capillary isfabricated from quartz.
 10. The spotting capillary of claim 1, whereinsaid spotting capillary is fabricated from glass.
 11. The spottingcapillary of claim 1, wherein said spotting capillary has an insidediameter (bore width) at the tip of less than about 100 μm.
 12. Thespotting capillary of claim 1, wherein said spotting capillary has aninside diameter (bore width) at the tip of less than about 50 μm. 13.The spotting capillary of claim 1, wherein said spotting capillary hasan inside diameter (bore width) at the tip that is less than the insidediameter away from the tip.
 14. In a microarray printer, a spottingcapillary according to any one of claims 1 through
 13. 15. A microarrayprinter comprising one or more spotting capillaries according to any oneof claims 1 through 13.